Project Summary More than 70 million people world-wide are infected with hepatitis C virus (HCV). Despite the development of effective new drugs to treat HCV, there were more new infections than cures in 60% of surveyed countries in 2016, including in the US. Therefore, development of a vaccine for HCV is essential for disease eradication. Early development of broadly neutralizing antibodies (bNAbs) is associated with spontaneous clearance of HCV in humans, but we cannot currently reproduce that protective immunity because we have not fully defined the epitopes targeted by bNAbs from individuals who cleared HCV infection, or the molecular features of anti- HCV antibodies that are critical for neutralizing breadth. We have developed a protocol which will allow us for the first time to sort HCV envelope protein (E2)-specific human B cells directly ex vivo. This is a critical first step that will allow us to develop single-cell antibody cloning methods for E2-specific bNAbs. In Aim 1, we will isolate E2-specific B cells from the blood of individuals who spontaneously cleared HCV infection, and then we will compare two monoclonal antibody single-cell cloning methods to identify the method that most efficiently isolates novel HCV-specific bNAbs. In Aim 2, we will select and engineer phenotypically distinct E2 protein antigens and antigen combinations. We will use these antigens to label and isolate HCV E2-specific B cells, followed by single-cell antibody cloning. We will characterize and compare the mAbs isolated using these different E2 antigens in order to identify the antigen or antigens that are most suitable for anti-HCV bNAb isolation. A vaccine for HCV is needed now more than ever. We now have an opportunity to isolate a large number and variety of anti-HCV bNAbs from humans who naturally cleared HCV infection, using methods that have been revolutionary in the study of bNAbs against influenza, HIV, and other viruses. Successful completion of this project will lead to identification of one or more efficient methods for single-cell antibody cloning of HCV- specific bNAbs, identification of E2 antigens or antigen combinations that favor isolation of exceptionally broad and potent neutralizing antibodies, and isolation of a large panel of novel anti-HCV bNAbs. Identification of the epitopes targeted and unique molecular features of these bNAbs will inform HCV vaccine development.